Intramembrane proteolysis plays a fundamental role in many biological and pathological processes. Intramembrane proteases thus represent promising pharmacological targets, but few selective inhibitors have been identified. This is in contrast to their soluble counterparts, which are inhibited by many common drugs, and is in part explained by the inherent difficulty to characterize the binding of drug-like molecules to membrane proteins at atomic resolution. Here, we investigated the binding of two different inhibitors to the bacterial rhomboid protease GlpG, an intramembrane protease characterized by a Ser–His catalytic dyad, using solid-state NMR spectroscopy. H/D exchange of deuterated GlpG can reveal the binding position while chemical shift perturbations additionally indicate the allosteric effects of ligand binding. Finally, we determined the exact binding mode of a rhomboid protease-inhibitor using a combination of solid-state NMR and molecular dynamics simulations. We believe this approach can be widely adopted to study the structure and binding of other poorly characterized membrane protein–ligand complexes in a native-like environment and under physiological conditions.

中文翻译：

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固态 NMR 和 MD 模拟的组合揭示了菱形蛋白酶抑制剂的结合模式

膜内蛋白水解在许多生物和病理过程中起着重要作用。因此，膜内蛋白酶代表了有希望的药理学靶点，但很少有选择性抑制剂被鉴定出来。这与它们的可溶性对应物形成对比，后者被许多常见药物抑制，部分原因是难以在原子分辨率下表征类药物分子与膜蛋白的结合。在这里，我们使用固态 NMR 光谱研究了两种不同抑制剂与细菌菱形蛋白酶 GlpG 的结合，GlpG 是一种以 Ser-His 催化二联体为特征的膜内蛋白酶。氘代 GlpG 的 H/D 交换可以揭示结合位置，而化学位移扰动另外表明配体结合的变构效应。最后，我们使用固态核磁共振和分子动力学模拟的组合确定了菱形蛋白酶抑制剂的确切结合模式。我们相信这种方法可以广泛用于研究其他特征较差的膜蛋白-配体复合物在类似天然环境和生理条件下的结构和结合。